Towards the development of a surface plasmon resonance assay to evaluate the glycosylation pattern of monoclonal antibodies using the extracellular domains of CD16a and CD64

Dorion-Thibaudeau, July; Raymond, Céline; Lattová, Erika; Perreault, Hélène; Durocher, Yves; Crescenzo, Grégory De

doi:10.1016/j.jim.2014.04.010

Cited by 26 publications

(60 citation statements)

References 35 publications

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“…Hardly any IgG binding to the low‐affinity Fcγ receptors and FcγRI was measured after DG, which is in agreement with results from others 12, 13, 15, 40. The binding to FcRn receptor is not or only moderately influenced by the glycan occupancy, as similar affinity at pH 6 was measured using deglycosylated IgG1 compared to the glycosylated reference IgG.…”

Section: Discussionsupporting

confidence: 92%

“…On the other hand, samples that contain no more than 2.5% of aggregates have no altered relative binding in our study, whereas Dorion‐Thibaudeau et al . 15 found that HMW levels of only 2% already affected the binding to FcγRIIIa in their assay. Due to the avidity effects of aggregates, the impact of a small fraction of dimers and higher oligomers in samples can alter binding to Fc receptors and can therefore not be neglected.…”

Section: Discussionmentioning

confidence: 96%

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Rapid screening of IgG quality attributes – effects on Fc receptor binding

Geuijen¹,

Oppers-Tiemissen

Egging

et al. 2017

FEBS Open Bio

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The interactions of therapeutic antibodies with fragment crystallizable γ (Fcγ) receptors and neonatal Fc receptors (FcRn) are measured in vitro as indicators of antibody functional performance. Antibodies are anchored to immune cells through the Fc tail, and these interactions are important for the efficacy and safety of therapeutic antibodies. High‐throughput binding studies on each of the human Fcγ receptor classes (FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb) as well as FcRn have been developed and performed with human IgG after stress‐induced modifications to identify potential impact in vivo. Interestingly, we found that asparagine deamidation (D‐N) reduced the binding of IgG to the low‐affinity Fcγ receptors (FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb), while FcγRI and FcRn binding was not impacted. Deglycosylation completely inhibited binding to all Fcγ receptors, but showed no impact on binding to FcRn. On the other hand, afucosylation only impacted binding to FcγRIIIa and FcγRIIIb. Methionine oxidation at levels below 7%, multiple freeze/thaw cycles and short‐term thermal/shake stress did not influence binding to any of the Fc receptors. The presence of high molecular weight species, or aggregates, disturbed measurements in these binding assays; up to 5% of aggregates in IgG samples changed the binding and kinetics to each of the Fc receptors. Overall, the screening assays described in this manuscript prove that rapid and multiplexed binding assays may be a valuable tool for lead optimization, process development, in‐process controls, and biosimilarity assessment of IgGs during development and manufacturing of therapeutic IgGs.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 96%

Rapid screening of IgG quality attributes – effects on Fc receptor binding

Geuijen¹,

Oppers-Tiemissen

Egging

et al. 2017

FEBS Open Bio

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“…The same observation has been made with bisecting N ‐Acetylglucosamine (GlcNac), most likely as it reduces the level of fucosylation (Umana et al ., ). On the same note, high levels of sialylation were shown to reduce ADCC activity (Scallon et al ., ), whereas Mabs presenting increased levels of galactosylation have been shown to interact with CD16a with higher affinity (Houde et al ., ; Dorion‐Thibaudeau et al ., ).…”

Section: Introductionmentioning

confidence: 97%

Biotinylation of the Fcγ receptor ectodomains by mammalian cell co-transfection: application to the development of a surface plasmon resonance-based assay

et al. 2015

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We here report the production of four biotinylated Fcγ receptor (FcγR) ectodomains and their subsequent stable capture on streptavidin-biosensor surfaces. For receptor biotinylation, we first describe an in-cell protocol based on the co-transfection of two plasmids corresponding to one of the FcγR ectodomains and the BirA enzyme in mammalian cells. This strategy is compared with a standard sequential in vitro enzymatic biotinylation with respect to biotinylation level and yield. Biotinylated FcγR ectodomains that have been prepared with both strategies are then compared by analytical ultracentrifugation and surface plasmon resonance (SPR) analyses. Overall, we demonstrate that in-cell biotinylation is an interesting alternative to standard biotinylation protocol, as it requires less purification steps while yielding higher titers. Finally, biotin-tagged FcγRs produced with the in-cell approach are successfully applied to the development of SPR-based assays to evaluate the impact of the glycosylation pattern of monoclonal antibodies on their interaction with CD16a and CD64. In that endeavor, we unambiguously observe that highly galactosylated trastuzumab (TZM-gal), non-glycosylated trastuzumab (TZM-NG), and reference trastuzumab are characterized by different kinetic profiles upon binding to CD16a and CD64 that had been captured at the biosensor surface via their biotin tag. More precisely, while TZM-NG binding to CD16a was not detected, TZM-gal formed a more stable complex with CD16a than our reference TZM. In contrast, both glycosylated TZM bound to captured CD64 in a stable and similar fashion, whereas the interaction of their non-glycosylated form with CD64 was characterized by a higher dissociation rate.

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“…FcγR 2aH, 2aR, 2b, 3aF, and 3aV were produced in 293-6E cells while FcγR 1a (CD64) was produced in CHO-3E7 cells according to previous work [36]. The human FcRn was also expressed in 293-6E cells by the co-transfection of the alpha subunit (p51) extracellular domain containing a TEV-cleavable C-terminal his-tag with B2-microglobulin in a 1:1 ratio.…”

Section: Fcγrs and Fcrnmentioning

confidence: 99%

“…The human FcRn was also expressed in 293-6E cells by the co-transfection of the alpha subunit (p51) extracellular domain containing a TEV-cleavable C-terminal his-tag with B2-microglobulin in a 1:1 ratio. Following purification as described in Dorion-Thibaudeau et al [36], the C-terminal his-tag was removed by TEV cleavage.…”

Section: Fcγrs and Fcrnmentioning

confidence: 99%

Asymmetric Fc Engineering for Bispecific Antibodies with Reduced Effector Function

et al. 2017

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Asymmetric bispecific antibodies are a rapidly expanding therapeutic antibody class, designed to recognize two different target epitopes concurrently to achieve novel functions not available with normal antibodies. Many therapeutic designs require antibodies with reduced or silenced effector function. Although many solutions have been described in the literature to knockout effector function, to date all of them have involved the use of a specific antibody subtype (e.g., IgG2 or IgG4), or symmetric mutations in the lower hinge or CH2 domain of traditional homodimeric monospecific antibodies. In the context of a heterodimeric Fc, we describe novel asymmetric Fc mutations with reduced or silenced effector function in this article. These heteromultimeric designs contain asymmetric charged mutations in the lower hinge and the CH2 domain of the Fc. Surface plasmon resonance showed that the designed mutations display much reduced binding to all of the Fc gamma receptors and C1q. Ex vivo ADCC and CDC assays showed a consistent reduction in activity. Differential scanning calorimetry showed increased thermal stability for some of the designs. Finally, the asymmetric nature of the introduced charged mutations allowed for separation of homodimeric impurities by ion exchange chromatography, providing, as an added benefit, a purification strategy for the production of bispecific antibodies with reduced or silenced effector function.

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Towards the development of a surface plasmon resonance assay to evaluate the glycosylation pattern of monoclonal antibodies using the extracellular domains of CD16a and CD64

Cited by 26 publications

References 35 publications

Rapid screening of IgG quality attributes – effects on Fc receptor binding

Rapid screening of IgG quality attributes – effects on Fc receptor binding

Biotinylation of the Fcγ receptor ectodomains by mammalian cell co-transfection: application to the development of a surface plasmon resonance-based assay

Asymmetric Fc Engineering for Bispecific Antibodies with Reduced Effector Function

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